Endochondral ossification is the process by which long bones, ribs, vertebrae and other skeletal elements form and grow during embryogenesis and after birth. During this process, preskeletal mesenchymal cells condense and differentiate into cartilaginous anlaga that provide the template and framework of the future skeleton. Within these incipient structures, the chondrocytes become organized in growth plates, undergo maturation and hypertrophy, and pave the way for endochondral bone formation. This critical multi- step process is recapitulated in healing of most bone fractures, but can also be usurped in pathologies such as heterotopic ossification. HO consists of formation of endochondral bone that forms and accumulates at ectopic sites and causes many health problems. One form of HO is common and non-genetic and is triggered by trauma, invasive surgeries and ensuing inflammation. A congenital, severe and often fatal form of HO is initiated by local flare-ups and is driven by activating mutations in ACVR1 encoding the type I bone morphogenetic protein (BMP) receptor ALK2. Up until recently, the acquired and congenital forms of HO were thought to follow the same pathogenic cascade given that they both involve an initial inflammatory stage and a subsequent endochondral process. However, recent studies by others suggested that congenital HO is caused by activin A, normally an antagonist of BMP/canonical signaling that in this condition, however, would interact with mutant ALK2 and elicit pSMAD1/5/8 signaling. If correct, the data would imply that genetic and non- genetic forms of HO are distinct processes distinguishable by the manner in which they are triggered at cell level and may require distinct therapies. Because we have long been working on the pathogenesis of genetic and non-genetic forms of HO and have a vested interest, we carried out preliminary studies to further assess those recent studies. We found that activin A is actually abundantly expressed in both non-genetic and genetic HO in mouse models, greatly stimulates chondrogenesis in wild type preskeletal mesenchymal cells, and promotes HO in wild type mice. These and other data lead to the central premise of this project that both genetic and non-genetic forms of HO involve, and are promoted by, activin A. The greater severity of genetic HO would result from greater responsiveness of mutant chondrogenic progenitors to activin A and unruly mutant ALK2 signaling and action, but both non-genetic and genetic forms of HO would be amenable to an anti-activin A-based therapy. Our Aims are: (1) To clarify the mechanisms of activin A action in chondrogenesis; (2) To determine activin A receptor composition and cell surface dynamics; and (3) To genetically test the roles of activin A in HO and its usefulness as a therapy target. We anticipate this project will provide novel insights into the pathogenesis of genetic and non-genetic forms of HO and also fundamental basic insights into the regulation of endochondral ossification. It will also provide a springboard toward an effective universal therapy for HO in both children and adults.